木聚糖酶EvXyn11TS耐热性与其N端二硫键的相关性分析

[目的]以糖苷水解酶11家族耐热木聚糖酶EvXyn11TS为研究对象,定点突变其编码基因Syxyn11,揭示EvXyn11TS耐热性与其N端二硫键的相关性.[方法]对不同来源的、与EvXyn11TS一级结构相似度较高的若干11家族木聚糖酶进行多序列同源比对,发现只有耐热的EvXyn11TS在其N端存在一个二硫键(Cys5-Cys32);运用分子动力学模拟预测该N端二硫键存在与否对木聚糖酶热稳定性的影响.以人工合成的Syxyn11为母本,采用PCR技术将其编码Cys5的密码子TGT突变为编码Thr5的ACT,构建去除了N端二硫键的突变酶(EvXyn11M)的编码基因Syxyn11M;分别将Syxyn11和Syxyn11M在毕赤酵母GS115中进行表达,并分析表达产物EvXyn 11 TS和EvXyn11M的温度和pH特性.[结果]酶学性质研究结果表明:EvXyn11M的最适温度Topt由突变前的85℃降至70℃;EvXyn11TS在90℃的半衰期t1/290为32 min,而EvXyn11M在70℃的半衰期t1/270仅为8.0 min.[结论]运用分子动力学模拟预测了N端二硫键对EvXyn11TS耐热性的重要作用,并通过定点突变验证之,为其它与耐热EvXyn11TS一级结构相似的、11家族常温高比活性木聚糖酶的耐热性改造提供了新的技术策略.     

Molecular Analysis of a Deletion Hotspot in the NRXN1 Region Reveals the Involvement of Short Inverted Repeats in Deletion CNVs

NRXN1 microdeletions occur at a relatively high frequency and confer increased risk for neurodevelopmental and neurobehavioral abnormalities. The mechanism that makes NRXN1 a deletion hotspot is unknown. Here, we identified deletions of the NRXN1 region in affected cohorts, confirming a strong association with the autism spectrum and other neurodevelopmental disorders. Interestingly, deletions in both affected and control individuals were clustered in the 5′ portion of NRXN1 and its immediate upstream region. To explore the mechanism of deletion, we mapped and analyzed the breakpoints of 32 deletions. At the deletion breakpoints, frequent microhomology (68.8%, 2–19 bp) suggested predominant mechanisms of DNA replication error and/or microhomology-mediated end-joining. Long terminal repeat (LTR) elements, unique non-B-DNA structures, and MEME-defined sequence motifs were significantly enriched, but Alu and LINE sequences were not. Importantly, small-size inverted repeats (minus self chains, minus sequence motifs, and partial complementary sequences) were significantly overrepresented in the vicinity of NRXN1 region deletion breakpoints, suggesting that, although they are not interrupted by the deletion process, such inverted repeats can predispose a region to genomic instability by mediating single-strand DNA looping via the annealing of partially reverse complementary strands and the promoting of DNA replication fork stalling and DNA replication error. Our observations highlight the potential importance of inverted repeats of variable sizes in generating a rearrangement hotspot in which individual breakpoints are not recurrent. Mechanisms that involve short inverted repeats in initiating deletion may also apply to other deletion hotspots in the human genome.     

Potato purple top phytoplasma‐induced disruption of gibberellin homeostasis in tomato plants

Phytoplasmas are phloem‐inhabiting, cell wall‐less bacteria that cause numerous plant diseases worldwide. Plants infected by phytoplasmas often exhibit various symptoms indicative of hormonal imbalance. In this study, we investigated the effects of potato purple top (PPT) phytoplasma infection on gibberellin homeostasis in tomato plants. We found that PPT phytoplasma infection caused a significant reduction in endogenous levels of gibberellic acid (GA₃). The decrease in GA₃ content in diseased plants was correlated with down regulation of genes responsible for biosynthesis of bioactive GAs ( GA20ox1 and GA3ox1) and genes involved in formation of GA precursors [geranyl diphosphate synthase (GPS) and copalyldiphosphate synthase (CPS)]. Exogenous application of GA₃ at 200 µmol L^?1 was able to restore the GA content in infected plants to levels comparable to those in healthy controls, and to attenuate the characteristic ‘big bud’ symptoms induced by the phytoplasma. The interesting observation that PPT phytoplasma‐infected plants had prolonged low expression of key GA biosynthesis genes GA20ox1 and GA3ox1 under GA deficiency conditions led us to hypothesise that there was a diminished sensitivity of the GA metabolism feedback regulation, especially GA biosynthesis negative feedback regulation, in those affected plants, and such diminished sensitization in early stages of infection may represent a central element of the phytoplasma‐induced disruption of GA homeostasis and pathogenesis.     

A high salt tolerant neutral protease from Aspergillus Oryzae: Purification, characterization and kinetic properties

A neutral high salt tolerant protease from Aspergillus oryzae CICIM F0899 which could be used for soy sauce production and other relevant applications under high-salt conditions was purified to homogeneity through ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography with overall recovery of 2%. Its molecular weight was estimated to be 50 kDa by SDS-PAGE. The optimum pH and temperature for activity of the extracellular protease of A. oryzae CICIM F0899 were shown to be between 7.0–9.0, and 50°C, respectively. The protease behaved high salt tolerance in 18% NaCl and retained 72% of initial activity after 14 days, indicating the high stability. The enzyme activity was inhibited by metal ions such as Al³⁺ and Ag⁺, and slightly activated by Mn²⁺ and Cu²⁺. A kinetic model incorporating the Debye-Hückel limiting law was proposed for A. oryzae CICIM F0899 protease hydrolysis of casein at ionic strength NaCl from 0.10 to 3.18 M. It was found that, with the higher ionic strength, the Michaelis constant K _m of the protease monotonically increased while the turnover number k _cat decreased in accordance with first order kinetic model. The high-salt tolerant protease has been demonstrated to be promising for the soy sauce production process.     

Flexibility is a mechanical determinant of antimicrobial activity for amphipathic cationic α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) are recognized as the potential substitutions for common antibiotics. Flexibility has been demonstrated to be a dominant on antimicrobial activity of an AMP, similar to the structural parameters such as hydrophobicity and hydrophobic moment as well as positive charge. To better understand the effect of flexibility on antimicrobial activity, we herein examined seventy-eight peptides derived from nine different species. Defined as a weighted average of amino acid flexibility indices over whole residue chain of AMP, flexibility index was used to scale the peptide flexibility and indicated to be a reflection of mechanical properties such as tensile and flexural rigidities. The results demonstrated that flexibility index is relevant to but different from other structural properties, may enhance activity against Escherichia coli for stiff clustered peptides or reduce activity against E. coli for flexible clustered peptides, and its optimum occurs at about − 0.5. This effect of flexibility on antimicrobial activity may be involved to the antimicrobial actions, such as stable peptide-bound leaflet formation and sequent stress concentration in target cell membrane, mechanically. The present results provide a new insight in understanding antimicrobial actions and may be useful in seeking for a new structure–activity relationship for cationic and amphipathic α-helical peptides.     

Imaging of jasmonic acid binding sites in tissue

Hormones regulate the mechanism of plant growth and development, senescence, and plants’ adaptation to the environment; studies of the molecular mechanisms of plant hormone action are necessary for the understanding of these complex phenomena. However, there is no measurable signal for the hormone signal transduction process. We synthesized and applied a quantum dot-based fluorescent probe for the labeling of jasmonic acid (JA) binding sites in plants. This labeling probe was obtained by coupling mercaptoethylamine-modified CdTe quantum dots with JA using N-hydroxysuccinimide (NHS) as a coupling agent. The probe, CdTe–JA, was characterized by transmission electron microscopy, dynamic light scattering, and fluorescent spectrum and applied in labeling JA binding sites in tissue sections of mung bean seedlings and Arabidopsis thaliana root tips. Laser scanning confocal microscopy (LSCM) revealed that the probe selectively labeled JA receptor. The competition assays demonstrated that the CdTe–JA probe retained the original bioactivity of JA. An LSCM three-dimensional reconstruction experiment demonstrated excellent photostability of the probe.     

Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers

Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds.     

Isolation and characterization of lactic acid bacteria from pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan

Lactobacillus pobuzihii is a novel species which has been previously found in pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan. However, the lactic acid bacteria (LAB) microflora in pobuzihi has not been studied in detail. In this study, LAB from pobuzihi were isolated, identified, and characterized. A total of 196 LAB were isolated; 79 cultures were isolated from the sample collected from a manufacturing factory, 38 from pobuzihi samples collected from 4 different markets, and 79 from 2 fresh cummingcordia samples. These isolates were characterized phenotypically and then divided into eight groups (A to H) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Lactobacillus plantarum was the most abundant LAB found in most samples during the fermentation of pobuzihi. On the other hand, Enterococcus casseliflavus and Weissella cibaria were, respectively, the major species found in the two fresh cummingcordia samples. A potential novel species or subspecies of lactococcal strain was found. In addition, seven L. plantarum and five W. cibaria strains showed inhibitory activity against the indicator strain Lactobacillus sakei JCM 1157^T. This is the first report describing the distribution and varieties of LAB existing in the pobuzihi during its fermentation process and the final product on the market.